Opportunistic infections by Pseudomonas aeruginosa are a major cause of death in cystic fibrosis patients and immune-compromised individuals. Quorum sensing is a cell-density-dependent gene-regulatory mechanism bacteria use to regulate the production of virulence factors and the formation of biofilms, both of which increase the morbidity of the infection. The virulence of P. aeruginosa, as well as other gram-negative human pathogens, requires an acyl-homoserine lactone (AHL) dependent quorum-sensing system. In addition to well-known gene-regulatory AHL receptors in P. aeruginosa, such as LasR and RhlR, there is an orphan AHL receptor, QscR, which lacks a cognate AHL synthase gene. QscR is a naturally occurring attenuator of P. aeruginosa virulence that can respond to AHLs produced by P. aeruginosa, as well as other species. A long-term goal of this work is to understand the molecular basis of quorum sensing gene regulation. In this proposal the mechanisms by which QscR responds to a broad range of AHLs will be elucidated. The central hypothesis is that QscR, through its ability to recognize a variety of AHLs produced by P. aeruginosa and other species, is a signal integrator that is allosterically controlled by signal binding to differentially regulate genes involved in P. aeruginosa quorum sensing. Determining the structural and biophysical basis for QscR AHL and promoter DNA recognition, as well as the AHL-dependent effects on the function of QscR will explain how QscR can respond to a broad range of AHLs to carry out virulence attenuation in P. aeruginosa. In the three Aims of this proposal, X-ray crystallography, biophysical, biochemical, and microbiology methods will be used to obtain new structural information about QscR and QscR-DNA complexes and to understand the structural and biophysical basis for QscR recognition of agonist and antagonist signals. This information will be validated in vivo and will guide the analysis of mutated QscR proteins in biological assays that assess QscR-dependent regulation of P. aeruginosa virulence. Together, these studies will provide the first complete structural and biophysical view of AHL receptor function in a human pathogen and bring new insights to quorum sensing gene regulation. PUBLIC HEALTH RELEVANCE: Current treatments of gram-negative bacterial infections will be greatly improved when the formation of bacterial biofilms and production of virulence factors can be prevented through the inhibition of quorum sensing. QscR is the first member of the AHL response factor family from a human pathogen, Pseudomonas aeruginosa that has proven amenable to a detailed analysis of the influence of signal binding on structure. This work is relevant to human health, because the structural basis of virulence modulation by QscR and QscR homologs will aid in the future development of virulence attenuating drugs, which may be used in the treatment of persistent, multi-drug resistant, and bioterrorism-related infections.